Use-Dependent Suppression of the Nicotinic Acetylcholine Receptor Response by the Proadrenomedullin N-Terminal 20-Amino Acid Peptide in Rat Locus Coeruleus Neurons

Abstract: The effects of the proadrenomedullin N-terminal 20-amino acid peptide (PAMP) on the nicotinic acetylcholine (ACh) receptor (nAChR)-mediated inward current were investigated in neurons acutely dissociated from the rat locus coeruleus using whole-cell recording under voltage clamp. Nicotine and cytidine mimicked the ACh response, whereas the maximal response to dimethyl-phenylpiperazinium was lower in amplitude compared with that to ACh. Nicotine-induced current ( I _nic) was suppressed more effectively by mecamylamine than by hexamethonium. In addition, neither atropine nor α-bungarotoxin affected the I _nic. PAMP reversibly and noncompetitively suppressed the peak amplitude of 10⁻⁴ M I _nic. PAMP concentrations for the threshold, half-maximal inhibition, and maximal inhibition of 10⁻⁴ M I _nic were 10⁻⁸, 2.6 × 10⁻⁷, and 10⁻⁵ M , respectively. The peak amplitudes of 10⁻⁴ M I _nic elicited at 2-min intervals showed a gradual decline in the presence of 10⁻⁷ M PAMP. This decline in the I _nic was independent of the period of PAMP pretreatment. The suppression of I _nic by PAMP did not show any voltage dependency at a holding potential ( V _H) of <0 mV, although the inhibitory effect was masked by the marked inward rectification of I _nic at a V _H of 0 mV. These results suggest that PAMP could thus be a unique endogenous peptide that antagonizes the nAChR in the CNS.     

Cholinergic innervation of the mouse superior cervical ganglion: light-and electron-microscopic immunocytochemistry for choline acetyltransferase

Summary The cholinergic innervation of the mouse superior cervical ganglion was investigated by means of immunocytochemistry using a well-characterized monoclonal antibody against choline acetyltransferase (ChAT). Immunopositive nerve fibers entered the superior cervical ganglion from the cervical sympathetic trunk. Light-microscopically, these fibers appeared to be heterogeneously distributed among the principal ganglion cells. The rostral part of the ganglion contained more ChAT-positive fibers then the middle or the caudal one. The axons branched several times before forming numerous varicosities. Most of the ChAT-stained fibers and varicosities aggregated in glomerula-like neuropil structures that were surrounded by principal ganglion cell bodies, whereas others were isolated or formed little bundles among principal neurons. None of the neurons or other cell types in the ganglion exhibited ChAT-positivity. ChAT-immunoreactive fibers disappeared from the ganglion 5 or 13 days after transection of the cervical sympathetic trunk. At the ultrastructural level, most axon terminals and synapses showed ChAT-immunoreactivity. An ultrastructural analysis indicated that immunostained synapses occurred directly on the surface of neuronal soma (1.8%) and dendritic shafts (17.6%). Synapses were often seen on soma spines (18.4%) and on dendritic spines (62.2%). All immunoreactive synapses were of the asymmetric type. The results provide immunocytochemical evidence for a heterogeneous cholinergic innervation of the ganglion and the principal neurons.     

Elektronenmikroskopische Untersuchungen zur funktionellen Morphologie des Tubero-hypophysären Systems der Ratte

Zusammenfassung 1. Die Gefäßfortsätze von ependymalen und subependymalen Zellen bilden in der subependymalen Zone und in der Zona interna des Ratteninfundibulum mit Nervenfasern kompakte neuro-gliöse Faserbündel, die sich in der Zona externa aufzweigen.2. In allen Zonen des Infundibulum kommen zwischen den Nervenfasern und den Gefäßfortsätzen zahlreiche neuro-gliöse Synapsen vor. In den Gefäßfortsätzen fällt die hohe Zahl an Mikrotubuli sowie die zahlreichen, vielgestaltigen Einschlüsse auf.3. In der subependymalen Zone sind die Interzellularspalten an bestimmten Stellen außerordentlich weit. Sie haben eine kanalartige Beschaffenheit und enthalten feine Bündel von Nervenfasern.4. Von den lateralen Anteilen des Infundibulum her erreichen Dendriten von Ganglienzellen des Nucleus infundibularis die Mitte des Infundibulum. In dieser Region sind axodendritische Synapsen anzutreffen.5. Morphometrische Analysen der Nervenfaserendigungen der Zona externa von Normaltieren zeigen, daß die prozentuale Verteilung der nach Granulagröße differenzierten Nervenfaserklassen für Mitte und Seite der Zona externa etwa gleich ist. Zwischen der Größe der Elementargranula und der Anschnittfläche der zugehörigen Nervenfasern besteht eine direkte Beziehung.6. Die Nervenfaserendigungen erreichen die Basalmembran des perikapillären Raumes fast ausschließlich im Bereich von gefäßwärts gerichteten Vorwölbungen der Zona externa. Das Ausmaß, in dem Nervenfasern im Vergleich zu den Gefäßfortsätzen von Ependymund Gliazellen den perivaskulären Raum erreichen, ist medial weitaus größer als lateral.7. Bei bilateral adrenalektomierten Ratten nimmt in bestimmten, vorwiegend lateral gelegenen Nervenfasern die Zahl und Größe der Elementargranula in Abhängigkeit von der Überlebensdauer zu. Dies dürfte auf eine verstärkte Synthese und Speicherung von Corticotropin-Releasing Factor in diesen Nervenfasern zurückzuführen sein. Gegenüber dem Normalbefund ist die neurohämale Kontaktfläche erheblich vergrößert. Der perivaskuläre Raum enthält zerfallene Nervenfaserteile, die durch Bindegewebszellen phagocytiert werden. Diese Veränderungen dürften durch eine unter Versuchsbedingungen verstärkte Wachstumstendenz der Nervenfasern in Richtung auf die Blutgefäße und durch eine Abschnürung der Nerven-faserendigungen ausgelöst werden.

---

Ultrastructural observations on the functional morphology of the tubero-hypophyseal system of the rat

Summary 1. In the subependymal and internal zones of the rat median eminence nerve fibres and vascular processes of ependymal and subependymal cells form neuro-glial bundles. They branch in the external zone.2. In all these three zones of the infundibulum numerous neuro-glial synapses are found between nerve fibres and vascular processes of glial cells. The vascular processes contain a high number of microtubules as well as polymorphous granular inclusions.3. In certain regions of the subependymal layer the intercellular spaces are enlarged. They form channel-like spaces containing small bundles of delicate nerve fibres.4. Nerve cells of the infundibular nucleus located in the lateral parts of the infundibulum send dendrites to the medial parts of the infundibulum. In this area axo-dendritic synapses are found.5. For morphometric analysis, the nerve fibres of the external zone were classified according to the diameter of their granules. It is shown that in the different regions of the external zone the distribution of the various types of nerve fibre is similar. Moreover it can be seen that a direct correlation exists between the size of the sectional plane of a given nerve fibre and the size of the granules it comprises.6. Nerve fibre endings abutting on the basement membrane of the pericapillary space are mostly found in bulb-like protrusions of the external zone. The extent to which nerve fibres reach the perivascular space—as compared with the vascular processes of ependymal and glial cells—is higher in the medial than in the lateral parts of the infundibulum.7. In bilaterally adrenalectomized rats the number and diameter of elementary granules increases in nerve fibres located laterally. This increase is directly related to the survival time and may be due to an enhanced synthesis and storage of Corticotropin-Releasing Factor in these nerve fibres. Compared with the findings in untreated animals the neurohemal contact area is significantly enlarged. The perivascular space contains degenerating nerve fibres which are undergoing phagocytosis by connective tissue cells. It is assumed that these alterations are due to the increased growth of nerve fibres towards the vessels of the Mantelplexus, and that, following adrenalectomy, this excessive growth leads to a pinching off of nerve fibres.

Teil einer Habilitationsschrift, die der Medizinischen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegen hat.     

Determination of the numerical density of perforated synapses in rat neocortex

Summary The numerical density and frequency of perforated synapses in the molecular layer of rat parietal cortex have been determined using 4 procedures in an attempt to overcome problems associated with the size and complex three-dimensional shape of perforated synapses. The following procedures were compared: A, single-section analysis; B, adjacent-section analysis; C, semi-serial-section analysis; and D, complete serial-section analysis. All procedures made use of an unbiased counting rule.Estimates of the numerical density of perforated synapses ranged from 0.06 to 0.27×10⁹ mm^-3, and that of all synapses (non-perforated and perforated) from 1.88 to 2.50×10⁹ mm^-3. The frequency of perforated synapses varied from 4.5 to 18.0%. Procedures B (adjacent-section analysis) and D (complete serial-section analysis), neither of which utilize assumptions regarding the shape of synapses, produced comparable results (numerical density of perforated synapses 0.19–0.27×10⁹ mm^-3, and of all synapses 2.24–2.45×10⁹ mm^-3; frequency of perforated synapses 8.6–10.9%). The frequency of perforated synapses appeared to be underestimated by procedure A (single section analysis; 4.5%) and overestimated by C (semi-serial section analysis; 18%).It is concluded that adjacent-section analysis is the most efficient and effective procedure for determining the numerical density and frequency of complex particles, such as perforated synapses. There is, however, no significant difference in the performance of this procedure compared with that of single-section analysis, for determining the numerical density of synapses in general. Nevertheless, inherent problems of bias within the single-section procedure make the adjacent section method the procedure of choice.     

The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors

We have found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) interacts with the beta subunits of the gamma-aminobutyric acid type-A receptor (GABA(A)R). BIG2 is a Sec7 domain-containing guanine nucleotide exchange factor known to be involved in vesicular and protein trafficking. The interaction between the 110 amino acid C-terminal fragment of BIG2 and the large intracellular loop of the GABA(A)R beta subunits was revealed with a yeast two-hybrid assay. The native BIG2 and GABA(A)Rs interact in the brain since both coprecipitated from detergent extracts with either anti-GABA(A)R or anti-BIG2 antibodies. In transfected human embryonic kidney cell line 293 cells, BIG2 promotes the exit of GABA(A)Rs from endoplasmic reticulum. Double label immunofluorescence of cultured hippocampal neurons and electron microscopy immunocytochemistry of rat brain tissue show that BIG2 concentrates in the trans-Golgi network. BIG2 is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABA(A)Rs. BIG2 is present in both inhibitory GABAergic synapses that contain GABA(A)Rs and in asymmetric excitatory synapses. The results are consistent with the hypotheses that the interaction of BIG2 with the GABA(A)R beta subunits plays a role in the exocytosis and trafficking of assembled GABA(A)R to the cell surface.     

Glutamatergic Synapses within the Framework of the “Chemical Soup” Concept: Relation to the Cognitive Activity

Modern concepts on the dependence of the efficacy of glutamatergic synaptic transmission on the subunit structure of glutamate (Glu) receptors and their chemical surroundings are described and discussed in the review. The above surroundings can significantly modulate the affinity of these receptors and/or change their number. Peculiarities of the effects exerted by different components of such surroundings (allosteric modulators, redox-active substances, some ions, hormones, etc.) on Glu receptors are analyzed. In the light of the chemical soup concept, the effects of Glu receptor ligands on the cognitive activity of experimental animals and of humans are discussed.     

Trophic factor effects on cholinergic innervation in the cerebral cortex of the adult rat brain

The cholinergic pathway ascending from the nucleus basalis magnocellularis (NBM) to the cortex has been implicated in several important higher brain functions such as learning and memory. Following infarction of the frontoparietal cortical area in the rat, a retrograde atrophy of cholinergic cell bodies and fiber networks occurs in the basalocortical cholinergic system. We have observed that neuronal atrophy in the NBM induced by this lesion can be prevented by intracerebroventricular administration of exogenous nerve growth factor (NGF) or the monosialoganglioside GM₁. In addition, these agents can upregulate levels of cortical choline acetyltransferase (ChAT) activity in the remaining cortex adjacent to the lesion site. Furthermore, an enhancement in cortical high-affinity³H-choline uptake and a sustained in vivo release of cortical acetylcholine (ACh) after K⁺ stimulation are also observed after the application of neurotrophic agents. Moreover, these biochemical changes in the cortex are accompanied by an anatomical remodeling of cortical ChAT-immunoreactive fibers and their synaptic boutons.     

Scanning electron-microscopic study on the three-dimensional structure of motor endplates of the slow (tonic) muscle fibers in the frog,Rana n. nigromaculata

Summary The three-dimensional organization of the motor endplates of the slow fibers of the rectus abdominis muscle in the Japanese meadow frog (Rana nigromaculata nigromaculata Hallowell) is visualized by use of a field-emission scanning electron microscope after removal of connective tissue components by HCl hydrolysis. Clusters of shallow oval depressions 1–3 m in diameter are seen in the postsynaptic membrane at intervals of about 150 m. On the surface of these depressions, a few low bulges of postsynaptic membrane are irregularly arranged. Terminal boutons, 1–3 m in diameter, occur along the length of nerve branches and terminals and fit into the shallow oval depressions of the postsynaptic membrane. The Schwann cells covering the terminal branches exhibit a simpler organization than those in twitch fibers.     

Characterization of Na+-Independent GABA and Flunitrazepam Binding Sites in Preparations of Synaptic Membranes and Postsynaptic Densities Isolated from Canine Cerebral Cortex and Cerebellum

The binding of [3H]GABA and [3H]flunitrazepam was performed with synaptic membranes and post-synaptic densities (PSDs) isolated from canine cerebral cortex and cerebellum. Two GABA binding sites were found with cerebral cortex membranes but only one with cerebellar membranes. PSDs isolated from these showed only single binding sites, with cerebellar PSDs exhibiting lower KD values and a larger concentration of sites than did cerebral cortex PSDs. In the case of flunitrazepam, only one binding site was found for all four preparations, with cerebellar PSDs having twice the concentration of sites of cerebral PSDs. Photoaffinity labeling of the flunitrazepam receptor in PSDs resulted in the binding to a 51,000 Mr protein in both cases, with cerebellar PSDs again showing an increased concentration over that found in cerebral cortex PSDs. Based on this work, and on earlier work of ourselves and of others, we conclude that both populations of isolated PSDs contain inhibitory sites, but that the intact PSDs in both preparations are derived from Gray type I, probably excitatory, synapses, and that the inhibitory sites are found in the broken-up material in the PSD fractions which are derived from Gray type II, probably inhibitory, synapses.     

Perioral synaptic connections and their possible role in the feeding behavior of Hydra

Electron microscopic observations of serially sectioned perioral neurons revealed a complex synaptic organization in which reciprocal synapses were observed for the first time in Hydra. Sensory cells had reciprocal synapses with each other and with ganglion cells, which in turn had reciprocal synapses with each other. A two-way chemical synapse with vesicles on both sides of the paramembranous densities was observed between ganglion cells; none was found between sensory cells. Ganglion cell axons participated in serial axo-axo-epitheliomuscular synapses. Two-cell pathways formed by direct sensory cell-nematocyte or neuromuscular synapses and three-cell pathways forming indirect sensory cell-ganglion cell-nematocyte or neuromuscular synaptic interconnections were found. It is possible that either simple direct changes in or direct effects on threshold stimuli could trigger both nematocyst discharge and/or muscular contraction and effect more complex intermediate pathways modulating feeding behavior. Each large epitheliomuscular cell enveloped from one to four sensory cells in the perioral region. The concentration of sensory cells around the mouth and their complex synaptic connections with each other and with ganglion and effector cells support our hypothesis for neural control of feeding behavior in Hydra.